Method of using two-stage nucleic acid reaction and detection tube

ABSTRACT

The present invention provides a method of using a two-stage nucleic acid reaction and detection tube. First, a reaction and detection tube is provided. Then a reaction is performed by using a liquid in the storing space of the second tube, following by putting the dipstick into the dipstick fixing space of the connector. The first tube, the second tube and the connector is assembled together, wherein the dipstick is placed both in the dipstick fixing space of the connector and the detection space of the first tube. The reaction and detection tube is rotated from bottom to top, thus making the liquid in the storing space of the second tube flow into the liquid collection space through the diversion unit and then contact the dipstick in the dipstick fixing space. A detection result is then observed from the dipstick.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.15/614,626 filed on Jun. 6, 2017, and included herein by reference.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to the field of biology, and moreparticularly, to a method of using a two-stage nucleic acid reaction anddetection tube which is to perform polymerase chain reaction (PCR)and/or nucleic acid detection and can be directly processed in the sametube without any liquid transfer.

2. Description of the Prior Art

Polymerase chain reaction (hereinafter referred to as “PCR”) is atechnology for rapid amplification of DNA, and its principle and themain operational steps may include: (a) denature: using a relativelyhigh temperature (90˜95° C.) for the double-stranded DNA dissociatinginto single stranded DNA, which is then used as a template forreplication; (b) primer annealing: when the temperature is lowered to apredetermined temperature, primers will be adhered to the correctpositions of the target genes; (c) primer extension: the reactiontemperature will be adjusted to 72° C., in combination of usingmagnesium ions as enzyme cofactors, the deoxy-ribonucleotidetriphosphate (hereinafter referred to as “dNTPs”) will be sequentiallyadhered to and after the primer by DNA polymerases, in accordance withthe base sequence of nucleotides on the template, so as to form asynthetic DNA fragment. By repeating the three-step process oftemperature oscillation, the number of the target gene can be doubled ineach repetition of the three-step process, thus can be 10⁹ times after40 cycles of the three-step process. The signal of the target gene cantherefore be amplified. Accordingly, the PCR detection technology isgenerally used for detecting molecular signals in clinical diagnosis,such as pathogen diagnosis, diagnosis of genetic disease, diagnosis ofcancer tumors, or the like. The RT-PCR technique which is derived fromPCR also has similar principle and application, therefore is widely usedin current techniques clinical diagnosis.

Devices used to perform PCR or RT-PCR reactions often include heatresistant plastics as the materials of the reaction tube. Theamplification of nucleic acid is achieved by using the thermostat metalto repeatedly increase and decrease the temperature for the tube so asto reach different temperatures in each three-step process. In currentsystem, the system with thermostat metal requires a relatively lagerspace, the entire temperature control system may occupy a larger spaceand heat capacity ratio. In addition, according to current practice ofoperation, one test required 30-35 cycles and the time required for thereaction is about two to three hours, consequently, most of the time ofthe process relies on waiting for the rise of the temperature or coolingmetal, making it difficult to reduce the reaction time.

In addition, the amplification of the target gene in PCR or RT-PCR isoften conformed by gel electrophoresis, which can separate the nucleicacid by its molecular size. After the PCR or PT-PCR process, the productcontaining amplified target gene (hereinafter “the product”) istransferred from the tube into a previously prepared gel well and sincethe nucleic acid contains negatively charge in neutral or alkalinesolution, it will move toward the anode wherein the moving speed isproportional to it molecular weight. By this process, it can be checkedif the amplification of nucleic acid successes or not. However, even thegel electrophoresis detection exhibits high accuracy, it still takesseveral hours to complete the PCR/RT-PCR and gel electrophoresis. Forthose diagnoses which are willing to obtain results in short time, itwould be not suitable to use PCR/PT-PCR to approach the diagnosis orinspection. In addition, when performing the gel electrophoresis, theproduct of PCR/RT-PCR needs to be transferred from the reaction vesselto the gel well, and the product is easily contaminated duringtransferring, which may result in false positive.

To shorten the reaction time, a technology that uses thermal convectionfor performing PCR has been developed (hereinafter “thermal convectionPCR”). This technology was first designed by Krishnan et al., T and ituses a cylindrical tube of the Rayleigh-Benard cell with two differentheating sources which are disposed at two corresponding sites of thetube. In general, the top level of the reagent is maintained at around atemperature of about 60° C., while the bottom temperature is about 95°C. By the temperature differences arising from the cylindrical cavitythrough the upper and lower end, it can drive the flow of fluid in thechamber, thus processing the PCR reaction. This embodiment may also beapplied to RT-PCR. From this, other technology derived from it withsimilar principle has been developed for commercial use, such as the useof isolated single point of heating technology called “insulatedisothermal polymerase chain reaction” (iiPCR), which performs the RCRreaction in a closed capillary in combination of three points heatingsources; or by using non-contact irradiation heating source, whichcontains heating point of the cylindrical tube closed loop design toachieve the effect for the PCR or RT-PCR. By using the thermalconvection process, PCR as well as RT-PCR will not need to usethermostat metal to be repeated temperature oscillation of three stepsof the reaction temperature, so it can save a lot of repeated heatingand cooling time, thus reducing the use of temperature control metal.

In order to avoid the product of the PCR/RT-PCR from beingcontamination, which would result in false positive, there are more andmore detection technologies used to replace original gelelectrophoresis, such as using a specific binding fluorescence chemicalcompound to combine the target gene. When a laser beam with appropriatewavelength is applied, the chemical compound will emit fluorescence, andit will be detected by the equipment. The intensity of the fluorescenceis proportional to the amount of the product of PCR/RT-PCR, so themethod can reach in-situ qualitative or even quantitative inspection,thus reducing the reaction temperature.

In another aspect, there is one common inspection technology that uses aspecific binding dipstick for the target gene to detect the product ofPCR/RT-PCR. The method includes using two different antigens that canspecifically bind antibody, for example, the first type of antigen isDIG or TexasR, and the second type of antigen is Avidin or FITC. Afterthe reaction, the product would contain these two types of antigens. Inaddition, one side of the dipstick is attached to an Ag gel, emulsionbeads or other coloring compounds, which can specifically bind theantibodies of the above-mentioned antigens, such as Biotin-anti-FITC orother specific binding protein; the other side of the dipstick isattached to a cotton pad; and some portions of the dipstick is coated tothe antibody of the first type of antigens such as anti-DIG, anti-TaxasRor other specific binding antibody. After the reaction, the product ofPCR or RT-PCR is transferred to the end of the lipstick that containsthe coloring compound, and the first type of antigen will specificallybind the first type of antibody to forma first typeantigen-antibody-coloring complex. The complex will then move toward theterminal end with cotton pad. When it moves at the part containing thesecond type of antibody, the second type of antigen will specificallybind the second type of antibody and thus exhibit colors. The user cantherefore obtain the result according to the colors. Since there areonly several minutes that are used to complete the dipstick detection,it can save more time in comparison to convention electrophoresismethod. In other situation, the antibody can be replaced by the nucleicacid probe depending on different requirement.

As described above, since the thermal convection PCR and the dipstickdetection method can save a lot of time comparing to conventionPCR/RT-PCR and gel electrophoresis method, it is widely used in theindustry in this field. However, there is still not found a detectiontube that can operate both thermal convection PCR and the dipstickdetection method in single tube. Conventional method is to complete thethermal convection PCR in one tube and then to transfer the reagent ontothe dipstick so as to carry out the entire detection method. However,since the product is difficult to take out from the tube, it is not suchconvenient to operate the system, and it also raises the risk ofcontamination.

Accordantly, there is still a need to have a two-stage detection tubethat can both perform polymerase chain reaction (PCR) and/or nucleicacid dipstick detection and can be directly processed in the same tubewithout any liquid transfer.

SUMMARY OF THE INVENTION

To solve the abovementioned problem, the present invention is directedto a two-stage nucleic acid tube. By using said tube, no additionalsteps of transferring chemical reagents are required. After performingthe convection PCR or PT-PCR, the first tube can directly connect thesecond tube with dipstick and the connector, the nucleic acid reactioncan be performed in one single device and the result can be checked bythe dipstick.

To achieve the above-mentioned object, according to one preferredembodiment of the present invention, the device includes a first tubeincluding a first connection portion and a detection space. Thedetection space is used for placing a dipstick with one cotton end; asecond tube including a second connection portion and a storing space,wherein the storing space is used to accumulate the sample of the targetgene and corresponding reagents. The connector includes a first portionand a second portion, which respectively connects the first connectionportion and the second connection portion, wherein the first portion ofthe connector comprises a diversion unit and a liquid collection space,the diversion unit can lead the liquid in the storing space to theliquid collection space, and the second portion comprises a dipstickfixing space for placing the dipstick, and the dipstick fixing space isconnected to the liquid collection space.

After performing the thermal convection PCR and/or the RT-PCR, thedipstick can be placed in the first tube and the connector in advance,and the first tube, the second tube and the connector are assembled toforma closed detection tube. After assembling the three components, theassembled device can be rotated from bottom to top with about 180degrees to make the reagents flow into the liquid collection spacethrough the diversion unit of the connector and thus contact thedipstick in the dipstick fixing space. By using the capillary action bythe dipstick with one end attaching cotton, the PCR product is deliveredfrom one end with the dipstick coated with coloring material toward theother end of the cotton dipstick. In this manner, the antigen carried bythe products can specifically combine the antibody which is coated onthe test paper, so the detection result can be observed through thedetection space. By the techniques set forth in the present invention,the object can be achieved by performing the convection PCR and/orRT-PCR to detect the products in one single device.

These and other objectives of the present invention will no doubt becomeobvious to those of ordinary skill in the art after reading thefollowing detailed description of the preferred embodiment that isillustrated in the various figures and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exploded drawing and assembly drawing of the detectiontube according to one preferred embodiment of the present invention.

FIG. 2A is an outward appearance of the first tube from one side.

FIG. 2B is a cross-sectional view taken along line BB′ in FIG. 2A.

FIG. 3A is an outward appearance of the first tube from another side.

FIG. 3B is a cross-sectional view taken along line AA′ in FIG. 3A.

FIG. 4 is a bottom view of the first tube.

FIG. 5A is an outward appearance of the second tube.

FIG. 5B is a cross-sectional view taken along line CC′ in FIG. 5A.

FIG. 6A is an outward appearance of the connector.

FIG. 6B is a cross-sectional view taken along line DD′ in FIG. 6A.

DETAILED DESCRIPTION

The following context and drawings illustrate the principles of thepresent disclosure according to one preferred embodiment of the presentinvention. As used herein, directional terms as may be used such as“horizontal,” “vertical,” “proximal,” “distal,” “front”, “rear”, “left,”“right,” “inner,” “outer,” “interior” and “exterior” relate to anorientation of the disclosed mixing device from the perspective of atypical user, and do not specify permanent, intrinsic features orcharacteristics of the device.

Please refer to FIG. 1. The reaction tube 1 in one preferred embodimentof the present invention includes a first tube 10, a second tube 30, anda connector 20 connecting the first tube 10 and the second tube 30.

Please refer to FIG. 1, FIG. 2A, FIG. 2B, FIG. 3A, FIG. 3B and FIG. 4.The first tube 10 in the previous embodiment is a transparency tube madeof poly-carbonate, which comprises a first connection portion 101, aholding portion 103, an observation window 102 disposed between thefirst connection portion 101 and the holding portion 103, and andetection space 104. In the present embodiment, the outward appearanceof the observation window 102 of the first tube 10 is a smooth and arcedplane. The holding portion 103 is located at one end of the first tube10, which has two corresponding surfaces to be clipped by the user'sfinger for observing the reaction result. At the other end correspondingto the holding portion 103, a first snap structure 105 is disposed inthe inner side of the first tube 10. In the present application, thefirst snap structure 105 includes at least an annular projection unit1051 for connecting the connector 20. The inner space of the first tube10 is the detection space 104 which is used to accommodate a dipstickcontaining one cotton end. The detection space 104 has an oblong shapein its cross-section, so the detection space 104 contains a pair ofparallel surfaces and a pair of curved surfaces. Thus, the outer surfaceof the first tube 10 and the inner surface walls of the first tube 10together forms a flat convex lens. When the dipstick is placed in thedetection space 104, the features shown on the dipstick can be enlargedand are easy to watch. Besides, when placing the dipstick, the endcontaining the cotton is positioned at the other side far from the firstsnap structure 105.

Please refer to FIG. 1, FIG. 5A and FIG. 5B. The second tube 30 is ahollow transparent tube having a portion with a greater inside diameterfor connection the connector 20 and a capillary portion with a smallerinside diameter. The second tube 30 has a second connection portion 301which has similar structure with that of the first connection portion101 of the first tube 10. A second snap structure 304 is formed insidethe second tube 30 and contains at least an annular projection unit 3041for connecting the connector 20. The storing space 303 inside secondtube 20 can accumulate the reagents required in the reaction.

As shown in FIG. 1, FIG. 6A and FIG. 6B, the connector 20 of the presentembodiment is an elastomer preferably made of silica gel so theconnector 20 has a hardness lower than the hardness of the first tube 10and the second tube 30. The connector 20 has a first portion 201 and asecond portion 202 to respectively connect the first connection portion101 of the first tube 10 and the second connection portion 301 of thesecond tube 30. In detail, both the first portion 201 and the secondportion 202 have a third snap structure 203 and in one preferredembodiment, the third snap structure 203 contains a plurality of annularprojection units 2031. When assembling the first tube 10, the secondtube 30 and the connector 20, the annular projection unit 1051 of thefirst tube 10 and the annular projection unit 3041 of the second tube 30can make the connector 20 slightly deformed, forming a compactassembling between the annular projection units of the first portion 201and the second portion 202, and the corresponding annular projectionunits 1051, 3041 of the first tube 10 and the second tube 30 so as toestablish a tightly integrated sealing structure.

Please refer to FIG. 6B, the first portion 201 of the connector 20includes a diversion unit 204 and a liquid collection space 205, and thesecond portion 202 includes a dipstick fixing space 206, which is a longchannel with an uniform caliber. The dipstick fixing space 206 has anoutlet 207 used as an entrance for putting the dipstick there through.The liquid collection space 205 has an inlet adjacent to the diversionunit 204 and an outlet adjacent to the dipstick fixing space 206,wherein the opening area of the outlet is greater than that of theinlet. By doing this, it can prevent inappropriate contact between thereagents and the dipstick and avoid the capillarity movement of thereagent toward the other side of the dipstick, which may result inunwanted reaction to the antibody coated on the dipstick. Further, itcan also prevent the reagent flowing into the detection space 104 in thefirst tube 10, which causes wrong interpretation of the result.

In the present embodiment, the diversion unit 204 is a diversion slope2041 inclining toward the liquid collection space 205. When the userconverts the detection tube 1 with 180 degrees, for example, turns thedetection tube in FIG. 1 upside down, the diversion slope 2041 can guidethe liquid in the second tube 202 to the liquid collection space 205.Since the liquid collection space 205 is a hollow chamber foraccumulating liquid and connected to the dipstick fixing space 206, whenthe liquid flows into the liquid collection space 205, the liquid wouldcontact the dipstick and soon processes the PCR/RT-PCR detectionreaction. Besides, as the inlet of the liquid collection space 205(referred to the interface between the liquid collection space 205 andthe diversion unit 204) is narrower than the outlet of the liquidcollection space (referred to the interface between the liquidcollection space 205 and the dipstick fixing space 206), the liquid canbe guided along the predetermined direction. In increasing the reactionspeed, the contact region of the liquid and dipstick is increased byforming the outlet of the liquid collection space 205 at the longerborder of the dipstick fixing space 206. By doing this, when the liquidflows out from the liquid collection space 206, it can contact thedipstick with a bigger area, so the reaction speed can be increased.

When using the detection tube, the to-be-analyzed target gene, thespecific binding antibody or other necessary are added into the secondtube 30 and a thermal convection PCR/RT-PCR. Thereafter, the product ofPCR/RT-PCR now contains the antigens called DIG or Avidin. Then, thedipstick that is coated by coloring material, specific binding antibodyand absorbent cotton in its terminal ends is placed into the dipstickfixing space 206 of the connector 20, following by assembly theconnector 20 and the second portion 301 of the second tube 30 togetherto make the dipstick fix in the storing space 303 of the second tube 30.In the present embodiment, the coloring material is gold (Au) gel, thespecific binding antigen is DIG in which the corresponding antibody isAnti-DIG, the other specific binding antigen is Avidin in which itscorresponding antibody is Biotin, and Biotin can bind Ag gel to form anAg gel-Biotin complex.

After the thermal convection PCR/RT-PCR, there is no need to remove thereagent in the second tube 30, instead, the connector 20, the first tube10 and the second tube 30 are assembled according to the structure inFIG. 1 thereby forming a completely compact detection tube 1.Thereafter, the detection tube 1 is turned 180 degrees, that is, makingthe structure if FIG. 1 upside down, so the reagents of the thermalconvection PCR/RT-PCR originally in the second tube would flow intoliquid collection space 205 and the dipstick fixing space 206 via theguide slope 204 as to contact the dipstick in the dipstick fixing space206. Since there is a cotton pad at one terminal end of the dipstick,the reagents of the PCR/RT-PCR will gradually move to the terminalcontaining the cotton because of the capillary action. When the movementof the reagent is processing, Avidin will specifically combine Biotin toform Avidin-Biotin-Ag gel complex, which will further move to the placewith Anti-DIG coating. Thus, it will combine with DIG and give colorsignals for detection. Consequently, the coloring can show the result ofdetection.

Those skilled in the art will readily observe that numerousmodifications and alterations of the device and method may be made whileretaining the teachings of the invention. Accordingly, the abovedisclosure should be construed as limited only by the metes and boundsof the appended claims.

What is claimed is:
 1. A method of using a two-stage nucleic acidreaction and detection tube, comprising: providing a reaction anddetection tube, comprising: a first tube, comprising a first connectionportion and a detection space; a second tube, comprising a secondconnection portion and a storing space; and a connector, comprising afirst portion and a second portion, wherein the first portion of theconnector comprises a diversion unit and a liquid collection space, andthe second portion comprises a dipstick fixing space, which connects theliquid collection space; performing a reaction by using a liquid in thestoring space of the second tube; putting the dipstick into the dipstickfixing space of the connector; assembling the first tube, the secondtube and the connector together by connecting the second portion of theconnector to the second connect portion of the second tube and byconnecting the first portion of the connector to the first connectionportion of the first tube, thereby forming the closed reaction anddetection tube, wherein the dipstick is placed both in the dipstickfixing space of the connector and the detection space of the first tube;and rotating the reaction and detection tube from bottom to top, thusmaking the liquid in the storing space of the second tube flow into theliquid collection space through the diversion unit and then contact thedipstick in the dipstick fixing space; and observing a detection resultfrom the dipstick.
 2. The method of using the two-stage nucleic acidreaction and detection tube according to claim 1, wherein the reactionis a thermal convection PCR or a thermal convection RT-PCR.
 3. Themethod of using the two-stage nucleic acid reaction and detection tubeaccording to claim 1, wherein liquid comprises a target gene andantigens.
 4. The method of using the two-stage nucleic acid reaction anddetection tube according to claim 3, wherein the antigens comprisedigoxigenin (DIG) or avidin.
 5. The method of using the two-stagenucleic acid reaction and detection tube according to claim 1, whereinthe dipstick is coated by a coloring material and antibodies.
 6. Themethod of using the two-stage nucleic acid reaction and detection tubeaccording to claim 5, wherein the coloring material is gold (Au).
 7. Themethod of using the two-stage nucleic acid reaction and detection tubeaccording to claim 5, wherein the antibodies comprise anti-digoxigenin(anti-DIG) or biotin.
 8. The method of using the two-stage nucleic acidreaction and detection tube according to claim 5, wherein the dipstickfurther comprises a cotton at one end in the detection space of thefirst tube.
 9. The method of using the two-stage nucleic acid reactionand detection tube according to claim 8, after rotating the reaction anddetection tube, the target gene is delivered along dipstick toward theend with the cotton by capillary action.
 10. The method of using thetwo-stage nucleic acid reaction and detection tube according to claim 1,wherein the detection result is observed from an observation windowshowing the dipstick.
 11. The method of using the two-stage nucleic acidreaction and detection tube according to claim 1, wherein an outperipheral contour of the first tube is a round tube and the detectionspace is an oblong space having two corresponding planar surfaces. 12.The method of using the two-stage nucleic acid reaction and detectiontube according to claim 1, wherein the second tube further comprises acapillary portion for placing the liquid.
 13. The method of using thetwo-stage nucleic acid reaction and detection tube according to claim 1,wherein the dipstick fixing space of the connector is an elongatedchannel with a uniform diameter.
 14. The method of using the two-stagenucleic acid reaction and detection tube according to claim 13, whereinthe liquid collection space comprises an outlet for leading the liquid,and the outlet is disposed at a longer side of the dipstick fixingspace.
 15. The method of using the two-stage nucleic acid reaction anddetection tube according to claim 1, wherein the liquid collection spaceis a chamber connecting the dipstick fixing space.
 16. The method ofusing the two-stage nucleic acid reaction and detection tube accordingto claim 1, wherein the liquid collection space has an inlet adjacent tothe diversion unit and an outlet adjacent to the dipstick fixing space,wherein the opening area of the outlet is greater than that of theinlet.
 17. The method of using the two-stage nucleic acid reaction anddetection tube according to claim 1, wherein the diversion unit is aguide slope inclining toward the liquid collection space for leading theliquid in the second tube toward the liquid collection space.
 18. Themethod of using the two-stage nucleic acid reaction and detection tubeaccording to claim 1, wherein the first connection portion comprises afirst snap structure and the second connection portion comprises asecond snap structure for connecting the connector.
 19. The method ofusing the two-stage nucleic acid reaction and detection tube accordingto claim 18, wherein the first snap structure or the second snapstructure comprises an annular projection unit for connecting theconnector.
 20. The method of using the two-stage nucleic acid reactionand detection tube according to claim 1, wherein a hardness of theconnector is less than a hardness of the first tube and a hardness ofthe second.